缺血后适应诱导线粒体保护的分子机制研究

Ischemic postconditioning, which refers to a single or a series of brief interference in the cerebral blood supply performed after a prolonged severe form of ischemic insult, has been reported to continuously improve neuronal functions after stroke. However, the molecular mechanisms responsible for such intraneous neuroprotection largely remain unknown. Previously, we found that a mitochondrial functional protein family of VDACs contributes to mitochondrial protection induced by ischemic postconditioning. Here, we propose that the ischemic postconditioning stables the expression of VDACs and then maintains the calcium homeostasis; on the other side, the ischemic postconditioning activates PI3K/Akt signaling, inhibits the activity of GSK-3beta and the subsequent disassembly of VDAC1-HK1 complex, which leads to anti-apoptotic effects. We will demonstrate such hypothesis in cultured neurons and ischemic animal models using the methods of biochemistry, molecular biology, pharmacology, and histology, etc. We will also investigate the transcriptional and post-transcriptional regulation mechanisms of VDACs following ischemic postconditioning. This study will reveals key signal molecules responsible for the neuroprotection and provides potential targets for the treatment of brain ischemia.

缺血后适应是指在缺血后再灌注期再次施以一个或多个相对短暂的缺血处理。研究发现，缺血后适应显著改善缺血后的神经功能，但其分子机制尚未阐明。申请人前期研究提示，线粒体功能蛋白VDACs介导缺血后适应的线粒体保护作用。本项目拟采用生物化学、分子生物学、药理学和组织学等方法，从整体和细胞水平证实本项目的设想：缺血后适应一方面可以稳定VDACs的表达，维持细胞内钙稳态；另一方面激活PI3K/Akt信号通路，抑制GSK-3beta的活性，稳定VDAC1-HK1信号复合体，从而发挥抗凋亡作用。本项目还将探明缺血后适应在转录和转录后水平调控VDACs稳定表达的分子机制，旨在发现神经保护作用的关键信号分子，为缺血性脑卒中神经保护药物的研发提供潜在的靶点。

缺血后适应明显改善缺血后神经功能。本项目旨在阐明缺血后适应触发神经保护的内源性信号网络和关键分子事件。研究发现：（1）SD大鼠全脑缺血后，海马CA1区组织中线粒体外膜蛋白VDAC1和VDAC3（VDAC1/3）水平显著下降，而缺血后适应维持VDAC1/3水平稳定；VDAC1/3敲减可以逆转缺血后适应神经保护作用。因此，VDAC1/3稳定是神经保护的关键环节。（2）体外双荧光素酶报告基因分析和在体研究显示，miR-7和miR-183/96靶向介导VDAC1/3的缺失；缺血后适应通过PI3K-Akt通路下调miR-183/96并稳定VDAC1/3；靶向miR-7和miR-183/96的核苷酸药物Anti-7和Anti-183减轻全脑缺血后的空间认知功能损害，还有利于局灶脑缺血后感觉运动功能的恢复；缺血性脑卒中病人外周血miR-7、miR-183和miR-96水平明显高于健康对照组。结果表明，miR-7、miR-183和miR-96是潜在的神经保护药物靶点，也是缺血性脑卒中快速诊断的潜在分子标志物。（3）缺血后海马组织ATP合成减少，缺血后适应使ATP恢复；Anti-7和Anti-183呈现相似的能量代谢稳态作用，VDAC1/3敲减可拮抗Anti-7和Anti-183的能量代谢稳态和抗凋亡效应；神经细胞中TP53介导脑缺血后VDAC1/3缺失，ATP产生减少进而促进神经损伤。结果表明，稳定VDAC1/3有益于线粒体能量代谢，从而改善神经功能。（4）缺血后适应和低剂量NMDA（5-40 nmol）后处理阻断MLK3-P38MAPKs信号通路，减轻Aβ神经毒性；非经典的MLK3-EPS8通路引起细胞骨架重构，可能成为潜在的药物靶点。总之，本研究提供了缺血后适应线粒体保护的分子机制，阐明稳定的VDAC1/3是能量代谢稳态和神经元存活的关键环节。缺血后适应保护具有广泛的临床意义，本项目为急性缺血性脑损伤、慢性退行性阿尔茨海默病的药物研发和临床治疗提供了新的思路。